Biomedical subjects
Publications and source records attributed to M Bengtsson.
During routine typing of a potential bone marrow donor, a new HLA DPB1 allele was identified. The new allele, officially named HLA DPB1*9001, differs from HLA DPB1*01011 in the second hypervariable region, where a single nucleotide substitution in position 191 changes the codon 35 from TAC to TTC with a predicted amino acid change from Tyr to Phe.
The presented "spot-on-a-chip" technology enables easy enrichment of samples in the low nanomolar (1-5 nM) range and provides a fast and reliable automated sample preparation method for performing matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis with high sensitivity and throughput. Through microdispensing, which allows accurate deposition of 60-pL droplets, dilute samples were enriched by making multiple droplet depositions in nanovials. The sample was confined to a defined spot area (300 x 300 microm), and multiple depositions increase the surface density of analyte in the nanovial, thereby providing detection of low attomole levels. The impact of the nanovial geometry with respect to the MALDI-TOF MS resolution for peptides deposited in the microfabricated silicon vials was investigated and the optimal geometry and size were determined. The spot-on-a-chip technology, that is, the combination of microdispensing, micromachined silicon nanovials and on-spot enrichment provides a signal amplification of at least 10-50 times as compared to an ordinary sample preparation. The linearity of the enrichment effect is shown by the analysis of a peptide mixture at the 5 nM level. The signal amplification provided by the spot-on-a-chip enrichment is demonstrated by the analysis of relevant biological samples, interleukin-8 from a spiked cell supernatant, and by successful protein identification of an excised spot from a high-sensitivity silver-stained two-dimensional electrophoresis gel separation.
The use of microfluidic components to create an analytical toolbox for the very rapidly growing field of proteomics is described. This toolbox provides novel generic analytical solutions that are highly adaptable for analysis of various biomolecules, ranging from high to low abundant. The components are fabricated using silicon micromachining and consist of a microchip immobilised enzyme reactor (microIMER), a piezoelectric microdispenser and high-density nanovial target plates. This microtechnology based platform interfaces matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI TOF-MS) to a wide range of upstream sample handling and/or analytical techniques. Examples of applications such as rapid on-line digestion (12 s) and sample preparation of proteins, interfacing to capillary liquid chromatography (100 attomol sensitivity), and in-vial chemistry on femtomol amounts of sample are presented.
Volatile compounds were collected from apple branches (Malus domestica) at different developmental stages, and the antennal response of codling moth females (Cydia pomonella) to these compounds was recorded by electroantennography coupled to gas chromatography. Presence of a range of terpenoid compounds, many of which had antennal activity, was characteristic for volatile collections from branches with leaves, and from small green apples. Nine compounds from branches with leaves and green fruit consistently elicited an antennal response: methyl salicylate, (E)-beta-farnesene, beta-caryophyllene, 4,8-dimethyl-1,3(E),7-nonatriene, (Z)3-hexenol, (Z,E)-alpha-farnesene, linalool, germacrene D, and (E,E)-alpha-farnesene. The bouquet emitted from flowering branches contained in addition several benzenoid compounds which were not found after bloom. Small green apples, which are the main target of codling moth oviposition during the first seasonal flight period, released very few esters. In comparison, fully grown apples released a large number of esters, but fewer terpenoids. The study of apple volatiles eliciting an antennal response, together with a survey of the seasonal change in the release of these compounds, is the first step toward the identification of volatiles mediating host-finding and oviposition in codling moth females.
The sex pheromone of the pea midge consists of 2-acetoxytridecane, (2S,11S)-diacetoxytridecane and (2S,12S)-diacetoxytridecane. The responses of male pea midges to the corresponding stereoisomers of (2S,11S)-diacetoxytridecane and (2S,12S)-diacetoxytridecane were tested in field trapping experiments and by electroantennographic recordings. When added at 20% of the pheromone component to the sex pheromone blend, the (2S,11R)- and (2R,11S)-stereoisomers of (2S,11S)-diacetoxytridecane, were shown to have a strong inhibitory effect on male attraction in the field. At the same dose, (2R,11R)-diacetoxytridecane, (2R,12R)-diacetoxytridecane, and meso-2,12-diacetoxytridecane, did not have a significant effect on male behavior. It was also shown that substitution of either (2S,11S)-diacetoxytridecane or (2S,12S)-diacetoxytridecane with the related stereoisomers reduced trap catches to the level of blank traps. The electroantennographic recordings showed similar dose-response curves for the pheromone components and the stereoisomers shown to have an inhibitory effect. It seems likely that male antennae have receptors for bothpheromone components and for inhibitory stereoisomers. Scanning electron microscopy and transmission electron microscopy of the antennae revealed three types of sensilla involved in chemoreception: sensilla circumfila, sensilla trichodea, and sensilla coeloconica. The sensilla circumfila and trichodea are both innervated by two sensory cells, whereas the sensilla coeloconica are innervated by four to five cells.
A new, previously unrecognized DPB1* allele, DPB1*8601, was found in a Swedish family. The new allele was carried on the common North European haplotype HLA A1-B8-DR3. Both individuals carrying the new allele were initially typed as clear DPB1*4601,*6601 but after family studies and further typing with allele-specific primers it was concluded that a new allele was present together with the common DPB1*0401. The new allele was investigated by direct sequencing of exon 2 in both forward and reverse directions employing intron primers combined with either an allele-specific sense or anti-sense biotinylated primer for bi-directional sequencing. The new allele is identical to DPB1*1701 in the five first variable regions. In the sixth region, however, DPB1*8601 carries the GGPM motif shared by several common alleles such as DPB1*0201 and 0401and 0402.
Peripheral T cell lymphomas (PTCL) have a poorer prognosis after conventional treatment than do high-grade B cell lymphomas. The place for high-dose therapy (HDT) with autologous stem cell support in these patients is still not clear. Forty patients, 10 women and 30 men, median age 41.5 years (range 16-61) with PTCL were treated with HDT and autologous stem cell support at The Norwegian Radium Hospital, Oslo, Norway and The University Hospital, Uppsala, Sweden, between February 1990 and September 1999. The histologic subtypes were: PTCL unspecified, 20 patients; intestinal, two patients; angioimmunoblastic (AILD), two patients; angiocentric, two patients and anaplastic large cell lymphoma (ALCL), 14 patients. All patients had chemosensitive disease and had received anthracycline-containing regimens prior to transplantation. At the time of HDT, 17 patients were in first PR or CR and 23 were in second or third PR or CR. Conditioning regimens were BEAM in 15 patients, BEAC in 14 patients, cyclophosphamide and total body irradiation (TBI) in eight patients, BEAC, without etoposide and TBI in one patient and mitoxantrone and melphalan in two patients. There were three (7.5%) treatment-related deaths. The estimated overall survival (OS) at 3 years was 58%, the event-free survival (EFS) 48% and the relapse-free survival (RFS) 56%, with a median follow-up of 36 months (range 7-100) for surviving patients. The patients with ALCL tended to have a better prognosis compared to those with other PTCL subtypes, OS 79% vs 44%, respectively. In conclusion, patients with chemosensitive PTCL who are failing to achieve CR with first-line chemotherapy or are in relapse can successfully be treated with HDT and autologous stem cell support.
Analysis of sex pheromone glands of the apple leafroller Bonagota cranaodes Meyrick by gas chromatography coupled with mass spectrometry or electroantennographic detection showed the presence of 14 structurally related acetates and alcohols of the chain length 10-18, including the main pheromone component (E,Z)-3,5-dodecadienyl acetate (E3,Z5-12Ac). Male antennae responded to the main compound, its Z,Z isomer, (E,Z) -3,5-tetradecadienyl acetate (E3,Z5-14Ac), and the monoenes (Z)-5-dodecenyl acetate (Z5-12Ac) and (Z)-9-hexadecenyl acetate (Z9-16Ac). Traps baited with a four-component blend of E3,Z5-12Ac, Z5-12Ac, E3,Z5-14Ac, and Z9-16Ac in a 100:5:5:100 ratio were significantly more attractive than the main compound alone. This improved trap lure is more suitable for monitoring population densities of B. cranaodes, and for detection of the onset of the seasonal flight period. A more complete pheromone blend is of importance also with respect to current attempts to develop mating disruption for control of this major pest of apple in Brazil.
The reducing oligosaccharides lactose and lacto-N-tetraose were reductively aminated with benzyloxycarbonylaminoaniline and sodium cyanoborohydride, followed by treatment of the resulting secondary amines with acetic anhydride. The resulting N-acetyl-N-(4-benzyloxycarbonylaminophenyl)-1-amino-1-deoxyaldi tol oligosaccharide derivatives were subjected to stepwise enzymatic elongation with various glycosyltransferases/nucleotide sugars. Purification of the products after each enzymatic step was conveniently performed by solid-phase extraction on silica gel C-18 cartridges. Two oligosaccharide derivatives (with sialyl Lewis a and sialyl dimeric Lewis x structures) were prepared. Conversion of the obtained derivatives into neoglycoproteins by the sequence hydrogenolysis, thiophosgene treatment, and protein coupling was carried out.
In plants, sesquiterpenes of different structural types are biosynthesized from the isoprenoid intermediate farnesyl diphosphate. The initial reaction of the biosynthesis is catalyzed by sesquiterpene cyclases (synthases). In Artemisia annua L. (annual wormwood), a number of such sesquiterpene cyclases are active. We have isolated a cDNA clone encoding one of these, amorpha-4,11-diene synthase, a putative key enzyme of artemisinin biosynthesis. This clone contains a 1641-bp open reading frame coding for 546 amino acids (63.9 kDa), a 12-bp 5'-untranslated end, and a 427-bp 3'-untranslated sequence. The deduced amino acid sequence is 32 to 51% identical with the sequence of other known sesquiterpene cyclases from angiosperms. When expressed in Escherichia coli, the recombinant enzyme catalyzed the formation of both olefinic (97.5%) and oxygenated (2.5%) sesquiterpenes from farnesyl diphosphate. GC-MS analysis identified the olefins as (E)-beta-farnesene (0.8%), amorpha-4,11diene (91.2%), amorpha-4,7(11)-diene (3.7%), gamma-humulene (1.0%), beta-sesquiphellandrene (0.5%), and an unknown olefin (0.2%) and the oxygenated sesquiterpenes as amorpha-4-en-11-ol (0.2%) (tentatively), amorpha-4-en-7-ol (2.1%), and alpha-bisabolol (0.3%) (tentatively). Using geranyl diphosphate as substrate, amorpha-4,11-diene synthase did not produce any monoterpenes. The recombinant enzyme has a broad pH optimum between 7.5 and 9.0 and the Km values for farnesyl diphosphate, Mg2+, and Mn2+ are 0.9, 70, and 13 microM, respectively, at pH 7.5. A putative reaction mechanism for amorpha-4,11-diene synthase is suggested.
Protein identification through peptide mass mapping by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has become a standard technique, used in many laboratories around the world. The traditional methodology often includes long incubations (6-24 h) and extensive manual steps. In an effort to address this, an integrated microanalytical platform has been developed for automated identification of proteins. The silicon micromachined analytical tools, i.e., the microchip immobilized enzyme reactor (mu-chip IMER), the piezoelectric microdispenser, and the high-density nanovial target plates, are the cornerstones in the system. The mu-chip IMER provides on-line enzymatic digestion of protein samples (1 microL) within 1-3 min, and the microdispenser enables subsequent on-line picoliter sample preparation in a high-density format. Interfaced to automated MALDI-TOF MS, these tools compose a highly efficient platform that can analyze 100 protein samples in 3.5 h. Kinetic studies on the microreactors are reported as well as the operation of this microanalytical platform for protein identification, wherein lysozyme, myoglobin, ribonuclease A, and cytochrome c have been identified with a high sequence coverage (50-100%).
Patients with Hodgkin's disease (HD) or large cell lymphomas who relapse after conventional chemotherapy have a poor prognosis without high-dose chemotherapy and stem cell transplantation (HDCT). Patients who relapse after HDCT have an extremely poor outcome. In the present study we describe four patients with relapsed HD (n=1) and large cell lymphomas (n=3) after HDCT. All had an aggressive clinical course before HDT. At relapse, however, they all have had prolonged remissions and/or spontaneous regressions on mild or no treatment. Possible explanations could be selection of less malignant clones or a lymphoma controlled by the immune system. The mechanisms are, however, unknown but warrant further studies of the molecular biology in these and similar cases.
Single sensillum recordings from Cydia pomonella male antennae showed three different types of receptor neurons. The most abundant type was most sensitive to the main pheromone compound (E,E)-8,10-dodecadienol, while its response to the geometric isomers E,Z, Z,E and Z,Z was comparable to a tenfold lower dose of (E,E)-8,10-dodecadienol. This neuron type also responded to the four behaviorally antagonistic isomers of (delta,delta)-8,10-dodecadienyl acetate, among which it was most sensitive to the E,E isomer. Cross-adaptation studies showed that these compounds were all detected by the same receptor neuron type. Receptor neurons specifically tuned to (E,Z) or (Z,Z)-8,10-dodecadienol were not found, although these two compounds are behaviorally active. A second type of receptor neuron responded to all isomers of (delta,delta)-8,10-dodecadienyl acetate and was most sensitive to the E,E isomer. This neuron type did not respond to any of the isomers of (delta,delta)-8,10-dodecadienol. A third receptor neuron type was highly sensitive to the plant compound alpha-farnesene. The finding that the receptor neuron type tuned to the main pheromone compound responded even to strong behavioral antagonists aids the interpretation of ongoing behavioral studies for the development of the mating disruption technique in codling moth.
Nucleosomes, the fundamental building blocks of chromatin, play an architectural role in ensuring the integrity of the genome and act as a regulator of transcription. Intrinsic properties of the underlying DNA sequence, such as flexibility and intrinsic bending, direct the formation of nucleosomes. We have earlier identified genomic nucleosome-positioning sequences with increased in vitro ability for nucleosome formation. One group of sequences bearing a 10-base pair consensus repeat sequence of TATAAACGCC had the highest reported nucleosome affinity from genomic material. Here, we report the intrinsic physical properties of this sequence and the structural details of the nucleosome it forms, as analyzed by footprinting techniques. The minor groove is buried toward the histone octamer at the AA steps and facing outwards at the CC steps. By cyclization kinetics, the overall helical repeat of the free DNA sequence was found to be 10.5 base pairs/turn. Our experiments also showed that this sequence is highly flexible, having a J-factor 25-fold higher than that of random sequence DNA. In addition, the data suggest that twist flexibility is an important determinant for translational nucleosome positioning, particularly over the dyad region.
Unrelated umbilical cord blood (UCB) as a source of haematopoietic stem/progenitor cells has been used for the first time in Sweden for allografting two boys (6 and 11 1/2 years old) with acute leukaemia in complete remission. Double class-II HLA-mismatch UCB units, from the Milan and New York cord blood banks, respectively, were used. Pre-transplant preparation consisted in fractionated total body irradiation in both cases, followed by cytoreductive high-dose cyclophosphamide for the 6-year-old with acute lymphocytic leukaemia (ALL) and fludarabine-melphalan for the 11 1/2-year-old with acute non-lymphocytic leukaemia (ANLL). Despite delayed haematopoietic recovery (probably due to HLA disparity) or reactivated cytomegalovirus infection in one boy, the immediate post-graft course was uneventful and extramedullary toxicity mild in both cases. Only transient acute graft-versus-host disease occurred, and complete donor chimerism was confirmed. Unfortunately, the out-come was unfavourable in both cases, the ANLL patient succumbing on day 96 due to pneumonia followed by multiorgan failure, and the ALL patient on day 140 due to combined medullar and central nervous system relapse. Although UCB transplantation needs further evaluation, it may contribute substantially to successful salvage procedures. Thus the introduction of cord blood banking in Sweden merits consideration.
In 30 patients with multiple myeloma who were scheduled for peripheral blood stem-cell transplantation, a quantitative analysis of the stem cells following enrichment by anti-CD34 was carried out. To detect the cells of the specific myeloma clone, polymerase chain reaction (PCR) was performed using unique allele-specific oligo primers for the immunoglobulin heavy chain rearrangement. The clonogenic cells before and after stem-cell enrichment, were quantified by a limiting dilution assay and a highly sensitive semi-nested PCR combined with a real-time quantitative PCR. In order to accomplish a statistically adequate end-point analysis, a large number of PCR analyses (40 per sample) were performed. By this technique the lowest detection limit observed was one myeloma cell per 106 cells. Myeloma cells were detected in 29/30 samples from the CD34-enriched fraction. The CD34 selection procedure resulted in a median 28-fold enrichment of CD34+ haemopoietic precursor cells. The stem-cell selection reduced the median concentration of clonal cells per million total cells by half, with a highly significant linear relationship between the number of myeloma cells before and after stem cell enrichment. The median depletion of clonal cells by the overall procedure was 2.15 log units, corresponding to a reduction of the total quantity of clonal cells reinfused into the patients by at least 99.3%. We conclude that CD34+ cell enrichment led to a reliable tumour cell depletion of the order of 2 log, which may not be sufficient since the total number of tumour cells in the leukapheresis product was 7.2 log (median).
The feasibility of mobilizing peripheral blood stem cells (PBSC) using anthracycline containing polychemotherapy and G-CSF on the first 50 patients randomized to the high-dose arm in the adjuvant SBG 9401 is investigated. The patients were treated with standard FEC (5-fluorouracil 600 mg/m2, epirubicin 60 mg/m2, cyclophosphamide 600 mg/m2) for two courses followed by a modified third FEC course with a C dose of 1200 mg/m2 supported with subcutaneous G-CSF (filgrastim) at 5 mg/kg followed by harvest around day 11. The mean yield of CD34+ cells per patient was 10.6x10(6)/kg (range 2.6-29.1). The side effects after the third course were low and only one patient developed an uncomplicated granulopenic fever. Our data indicated a correlation between number of transfused CD34+ cells and days to neutrophil and platelet recovery. In conclusion, the modified FEC regimen followed by G-CSF is a feasible method for PBSC mobilization in the adjuvant setting.
The aim of the present study was to investigate the N-terminal part (the translated part of exon 1) of the human thyrotropin receptor (TSHR) for the presence of mutations. Patients with Graves' disease (n = 160) and healthy controls (blood donors; n = 140) were screened using single-stranded conformational polymorphism (SSCP) in combination with restriction enzyme digestion for the two previously known mutations in this part of the receptor, viz. D36H and P52T TSHR-variants. We did not find any novel mutation in this region. However, D36H and P52T variants were found both in the TSHR of Graves' patients and in the healthy controls. The overall frequency of the D36H-receptor variant was 5.0% (15/300) and of the P52T-receptor, 7.3% (22/300). There was no major difference in the frequency for either of the TSHR alleles between the 2 groups. Thus, these 2 polymorphic variants of the TSHR seem to occur in a relatively high frequency in the population.